Project Summary The long range goals of this proposal are to define molecular mechanisms by which mechanical forces experienced by cells influence growth control pathways, and to determine where, when and how these mechanisms are employed in vivo to modulate organ size. They build on the discovery in Drosophila of a biomechanical mechanism for modulating the Hippo pathway, which is a key regulator of growth and cell fate in all animals, including humans. This mechanism is triggered by cytoskeletal tension-dependent recruitment of the Ajuba family LIM protein (Jub) to ?-catenin at adherens junctions. Jub then recruits and inhibits the key Hippo pathway kinase, Warts, which leads to increased activity of Yorkie, a transcription factor of the Hippo pathway. Our studies will enhance understanding of how cells respond to their mechanical environment, how biomechanical signaling is integrated with biochemical signaling, and how biomechanical signaling modulates growth and cell fate decisions. The first aim proposes studies to identify components of this pathway in mammalian cells and to determine the contribution of this pathway to the regulation of Hippo signaling in different mechanical environments. We also propose to develop methods to visualize the activity of this pathway in live mice. The second aim proposes genetic and biochemical approaches to investigate the molecular mechanism by which forces are actually sensed and transmitted to the Ajuba and Warts family proteins. The third aim investigates the relationship between the influence of cytoskeletal tension on Ajuba family proteins, and its influence on ?-catenin, through a combination of biochemical, imaging, and genetic approaches, and will employ both Drosophila and cultured cell models. These studies investigate fundamental processes that cells use to respond to their mechanical environment. The results of these studies will be relevant to understanding both normal development and physiology, and to disease states associated with either insufficient or excess growth. As inappropriate growth during development results in organs that are incorrectly sized or shaped, it can cause birth defects. Controlling organ growth is also important for understanding how stem cells can be used to repair or replace damaged organs, which is a goal of regenerative medicine. Additionally, the inability to limit growth in mature organisms results in cancer. Cancers in a wide variety of organs have been associated with inactivation of Hippo signaling, including liver, kidney, skin, brain, intestine, lung, ovary, breast, and prostate. Understanding the regulation of Hippo signaling is thus relevant to a range of human health issues, including birth defects, cancer, and regenerative medicine.